Assays for the detection and/or quantification of analytes exist in many different forms and formats. In many cases, the amount of the analyte sought to be detected in a sample is not great enough for direct detection or quantification. In such cases, a secondary molecule able to bind to or interact with the analyte must be used to indicate the presence or amount of analyte in a given sample. Unless the secondary molecule is directly detectable, the secondary molecule must be conjugated with a label which will be detected when the secondary molecule binds the analyte. For the purposes of the present application, such secondary molecules able to bind or interact with analytes will be referred to as "binding partners" or "probes". A non-exclusive list of analytes sought to be detected are antibodies, proteins, cell-surface receptors, cytokines, hormones, antigens, nucleic acids, metals, molecular complexes such as polymeric arrangements of proteins or other macromolecules, and the like. Likewise, binding partners for the detection of such analytes may include, without limitation: antibodies, proteins, antigens, haptens, nucleic acid probes, chelating agents, enzymes, enzyme substrates, and analogs of these.
One commonly used assay format is the enzyme-linked immuno-absorption assay (ELISA). In this assay format the analyte is contacted with a primary antibody able to bind at least one domain or "target region" thereon. After the excess antibody is washed free of the resulting analyte: antibody complex, the primary antibody is contact with an enzyme-labeled secondary antibody to which it will specifically bind. The sample is then given a chromogenic enzyme substrate, and incubated under conditions favoring enzyme-mediated reaction of the substrate. The resulting colored product and its intensity after a given reaction time are indications of the presence and amount, respectively, of the analyte originally present in the sample. Illustrative examples of enzymes used in such assay methods are .beta.-galactosidase, acid phosphatase, and alkaline phosphatase; a non-exhaustive list of enzyme substrates for use with such enzymes include x-gal (5-bromo-4-chloro-3-indolyl-.beta.-D-galacto-pyranoside) and p-nitrophenyl phosphate. Other such enzymes and substrates are well known by those of skill in the art. Variations of this assay method exist; for example, the primary antibody may be linked to an enzyme thus eliminating the secondary antibody step. Nevertheless, these assay methods feature common steps involving contacting of the analyte with a labeled binding partner and subsequent detection of the analyte-bound label as an indication of the presence or amount of analyte.
While ELISA utilizes an enzyme label which is indirectly detected, other methods exist for the direct detection of labeled binding partner. Thus, Campbell, et al., U.S. Pat. No. 4,496,958 describes chemiluminescent acridinium labeling compounds for use in labeling binding partners in multiple assay formats; this patent is incorporated by reference as part of the present application. Additionally, other labeling compounds such as radionuclides, fluorescent, bioluminescent, phosphorescent, luminescent, chemiluminescent, or electrochemiluminescent compounds, chromophores, and dyes are known in the art and are commonly used as labeling agents in a variety of assay formats, both direct and indirect, including immunoassay and nucleic acid hybridization assays.
In addition to distinctions between assays based on the type of analytes to be detected, the type of binding partner with which the analyte binds, and the type of label used, assays can also be classified according to whether the method involves the immobilization of the analyte, or the analyte: binding partner complex. In the most common assay format, known as a "heterogeneous" or biphasic assay system, a probe is allowed to bind its analyte--usually under conditions of probe excess. Either the analyte molecule or the probe molecule may be immobilized to a solid support, thus causing the resulting probe: analyte complex to become immobilized--alternatively, and preferably, the complex may be immobilized following its formation in the liquid phase. After probe: analyte complexes have been immobilized, the excess uncomplexed probe molecules are washed away. If the probe molecules are directly labeled, the label may now be detected as an indication of the presence of analyte. In a variation of this format, probe analyte complexes may also be separated from free probe, by means such as gel filtration chromatography, electrophoresis, electrofocusing, and other separation methods based on size or charge of the probe: analyte complex.
Alternatively, and more rarely, an assay may be designed to take place wholly in a single phase without a step resulting in the physical separation of probe: analyte complex from free probe. Such assay methods are termed "homogeneous" assays. In such methods, usually either the analyte: probe complex, or the free probe is altered after formation of the complex to permit the separate detection of analyte in the presence of the free probe. One such way of differentiating free probe from probe-bound analyte involves alteration or selective inactivation of the label joined to the probe rather than the free probe molecule itself. Arnold, et al., U.S. Pat. No. 5,283,174, describes homogeneous methods employing a oligonucleotide probe joined to a label which is capable of selective inactivation or alteration based on whether the labeled probe is bound to its target or not. These methods may be used in a single tube without the need for washing or decanting. This patent enjoys common ownership with the present application, and is incorporated by reference herein.
Assay methods exist which may utilize aspects of both homogeneous and heterogeneous assays. These methods may, for example, employ a single phase selective alteration of the probe or the probe: analyte complex followed by a physical separation step to further decrease the level of background in the assay. Such a "hybrid" assay format is described as one aspect of the multiple analyte assay described in Nelson et al., U.S. Pat. No. 5,658,737 which is incorporated by reference herein.
Regardless of the assay format used, a number of factors exist in all assays which can limit their sensitivity and the range of possible analyte concentrations that can be accurately detected or measured. One such factor is the level of background present in the assay. "Background" is a term used to describe probe or label in the assay which is not bound specifically to analyte and which may mask positive results at low analyte levels. Thus, for example, in a heterogeneous assay, background may be provided by a small amount of probe which is not removed during the physical separation of probe: analyte complex from free probe. If the probe is labeled, this small amount of probe will be detected as a residual level of detectable signal. In a homogeneous assay, background may be provided by the inability to totally alter all free probe or, probe-linked label molecules. In either case, a small level of signal, commonly between 0.001% and 10% of the total signal, more commonly between about 0.01% and 1%, is present as a "baseline" below which results cannot be relied on for accuracy. Thus, the level of background inherent in a particular assay format limits the lowest amount of analyte which can be detected and/or measured.
The phenomenon of residual background in an assay plays a part in limiting the "dynamic range" of that particular assay. By "dynamic range" is meant a linear or predictably accurate correspondence between the level of analyte present in the sample to be assayed and the amount of signal obtained from the label used to indicate the analyte's presence. It is readily apparent to those of skill in the art that the dynamic range of an assay cannot extend below the level of background contributed by the detection of non-specific label, thus the higher the background, the more the dynamic range is limited. Moreover, if high amounts of analyte are to be detected, correspondingly high amounts of probe have to be used, which leads to higher backgrounds.
Other factors may contribute to limitations on the extent of a particular assay's dynamic range. A major additional factor is often the maximum amount of signal able to be read or reported by the label detection device or instrument to be used in the assay. Thus, if a given instrument can only accurately read up to, for example, one million counts per second and the sample yields 2 million counts per second, the extra one million counts are not being reported by the instrument, and the upper extent of the assay dynamic range is half of what is necessary to accurately quantify the sample.
Additionally, instruments used to detect the labeled probe: analyte complexes may have inherent electronic "noise", which is also commonly termed "background", and which can also contribute to limitations on the accuracy of the detection of the analyte. While improvements to, and optimization of an instrument's electronic signal-to-noise ratio are possible, the noise obtained in a specific instrument combines with the inherent assay background described above to further limit the ability to detect or quantify analytes across a wide range of possible concentrations. In order to overcome these limitations, it is currently necessary for workers to obtain multiple samples from a single source, or to make serial dilutions of a sample to test for the presence of an unknown amount of analyte.
There is, therefore, currently a need in the art for methods and compositions for detecting and/or quantifying analytes of every kind in a single tube without the need for sample dilutions or duplicate samples. Preferably, such methods and compositions would involve a single addition of detection probes from which possible analyte concentrations differing by orders of magnitude can be detected. Such methods should also generally be independent of the analyte type, probe type, label type, and instrument to be used for the detection of analyte.